Accepted Manuscript Title: Colloidal gold nanoparticle conjugates of gefitinib Author: Anh Thu Ngoc Lam Jinha Yoon Erdene-Ochir Ganbold Dheeraj K. Singh Doseok Kim Kwang-Hwi Cho So Yeong Lee Jaebum Choo Kangtaek Lee Sang-Woo Joo PII: DOI: Reference:
S0927-7765(14)00444-5 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.08.021 COLSUB 6586
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
5-1-2014 15-8-2014 17-8-2014
Please cite this article as: A.T.N. Lam, J. Yoon, E.-O. Ganbold, D.K. Singh, D. Kim, K.-H. Cho, S.Y. Lee, J. Choo, K. Lee, S.-W. Joo, Colloidal gold nanoparticle conjugates of gefitinib, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.08.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
an
us
cr
ip t
Colloidal gold nanoparticle conjugates of gefitinib
a a a b b Anh Thu Ngoc Lam , Jinha Yoon , Erdene-Ochir Ganbold , Dheeraj K. Singh , Doseok Kim , Kwang-
a
d
M
Hwi Choc, So Yeong Leed, Jaebum Chooe, Kangtaek Leef, Sang-Woo Joo a ,*
Department of Chemistry, Soongsil University, Seoul 156-743 Korea Department of Physics, Sogang University Seoul 121-742 Korea
c
School of Systems Biomedical Science, Soongsil University, Sangdo-dong, Dongjak-gu, Seoul, Korea
d
School of Veterinay Science, Seoul National University, Seoul 156-743, Korea
e
Department of Bionano Engineering, Hanyang University, Sa-1-dong 1271, Ansan 426-791, South Korea
f
Ac ce p
te
b
Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Republic of Korea
*
Corresponding author E-mail address: [email protected]
1
Page 1 of 23
Abstract
Gefitinib (GF) is a US Food and Drug Administration-approved epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor for treating the lung cancers. We fabricated colloidal gold nanoparticle
ip t
(AuNP) conjugates of the GF anticancer drug by self-assembly to test their potency against A549, NCIH460, and NCI-H1975 lung cancer cells. GF adsorption on AuNP surfaces was examined by UV-Vis
cr
absorption spectra and surface-enhanced Raman scattering. Density functional theory calculations were
us
performed to estimate the energetic stabilities of the drug-AuNP composites. The N1 nitrogen atom of the quinazoline ring of GF was calculated to be more stable than the N3 in binding Au cluster atoms.
an
The internalizations of GF-coated AuNPs were examined by transmission electron and dark-field microscopy. A cell viability test of AuNP-GF conjugates with the EGFR antibody exhibited much
M
higher reductions than free GF for A549, NCI-H460, and NCI-H1975 lung cancer cells after treatment
te
d
for 48 h.
Ac ce p
Keywords: Lung cancer cells; Gold nanoparticle conjugates; Gefitinib; Cell viability; Surface-enhanced Raman scattering
2
Page 2 of 23
1. Introduction Recently, understanding and controlling interfacial phenomena in biotechnology has become increasingly important in both fundamental research and industry [1]. Noble metal nanoparticles (NPs) have been introduced as novel drug delivery systems for anticancer therapy [2-5]. Gold nanoparticles
ip t
(AuNPs) after binding with the drugs may overcome antibiotic drug resistance [6]. AuNPs are known to be less toxic and can possibly be used in a hospital environment for immunotherapy [7]. Surface-
cr
enhanced Raman scattering (SERS) has been used to examine the adsorption structures in bioconjugate
us
chemistry [8,9]. Quantum mechanical calculations can be helpful to better understand the adsorption structures of self-assembled monolayers on metal cluster atoms [10].
an
Lung cancer is one of the leading causes of cancer-related deaths worldwide [11]. Expressed epidermal growth factor receptor (EGFR) can be utilized in lung cancer as a prime candidate for the development
M
of targeted therapeutics. Targeting EGFR is thus one of the most promising strategies for lung cancer therapy. In association with EGFR, tyrosine kinase inhibitor (TKI) has emerged as an effective cure for
d
some patients [12-16]. An EGFR-targeting small-molecule inhibitor, gefitinib (GF), received approval
te
from the US Food and Drug Administration as treatment for lung cancer patients, because it specifically
Ac ce p
targets the tyrosine kinase activity of EGFR, is one of the most popular and commercially available drugs to treat lung cancers [17-19].
Despite previous studies of nanoparticle conjugates with anticancer drugs, there have been no reports on the interfacial behavior of GF on AuNPs. GF may coordinate with AuNP surfaces via the quinazoline ring. In this work, we fabricated an AuNP conjugate of GF by self-assembly. Density functional theory (DFT) calculations were introduced to predict the plausible geometries of GF on an Au atom cluster. The adsorption behavior of GF on AuNP surfaces was studied mainly by means of SERS. A cell viability test was performed to examine their potency against A549, NCI-H460, and NCIH1975 lung cancer cells. Our work may be potentially useful in the application of AuNP conjugates in lung cancer therapy.
3
Page 3 of 23
2. Experimental section 2.1. Chemicals HAuCl4 and citrate were purchased from Sigma-Aldrich (St. Louis, USA). GF and mouse monoclonal anti-EGFR antibody (catalogue number: Ab62, concentration 1.2 mg/mL) were purchased from Selleck
ip t
Chemicals (Houston, USA) and Abcam (Cambridge, UK), respectively. Dimethyl sulfoxide (DMSO) was purchased from Daejung (Siheung, Korea). The (ortho-pyridyl) disulfide-poly(ethylene glycol)-N-
cr
hydroxysuccinimidyl ester (OPSS-PEG-NHS) linker was purchased from Creative PEGWorks (Winston
us
Salem, USA).
an
2.2. Preparation of nanoparticles
Citrate-modified AuNPs were prepared according to the previous procedure [20]. The AuNP-GF
M
sample was prepared by adding 10 μL of GF to 1 mL of AuNPs via a self-assembly process. The mixture was centrifuged at 10000 rpm for 5 min. To estimate the loading efficiency of GF onto AuNPs,
d
the UV band of 1 mL of supernatant solution was compared with that of 1 mL of the precipitated
te
solution redispersed in DMSO. The loading efficiency of AuNPs-GF (10-5 M) in DMSO was estimated
Ac ce p
to be 26-46 %, depending on the solvents and concentrations. To attach the EGFR targeting moiety to AuNPs, we suspended 10 µL of antibody (1mg/mL) in 90 µL of NaHCO3 (pH 8.5) according to the previous report [21]. The OPSS-PEG-NHS linker was prepared with a concentration of 1mg/mL (1 mg of OPSS in 1 mL of NaHCO3 of pH 8.5). Then, 100 µL of antibody and 100 µL of OPSS-PEG-NHS in a ratio of 1:1 was mixed and maintained at 4 °C for approximately 3 h. Subsequently, 1mL of AuNP-GF was added to 200 µL of antibody-OPSS-PEG-NHS and maintained at 4 °C overnight. The mixture solution was then centrifuged at 10,000 rpm for 5 min. After collecting the precipitate, it was dissolved again in 1 mL of DMSO solution. AuNPs-GF-antibody was prepared with a GF concentration of 10-4 M.
2.3. Equipment and characterization methods
4
Page 4 of 23
This amount of AuNP conjugates was diluted to 10-5 M by DMSO solvent and used for the CCK experiment. The UV-Vis absorbance spectrum of the surface plasmon band from the AuNP solution was obtained using a Scinco Mecasys 3220 spectrophotometer. The size distributions of AuNPs were checked using either a JEOL JEM-3010 or JEM-1010 transmission electron microscope (TEM). Raman
ip t
spectra equipped with dark-field microscopy (DFM) were obtained using a Renishaw 1000 microRaman spectrometer as in the previous report [22]. The excitation wavelength was 632.8 nm. The spontaneous
cr
scattering from the sample was dispersed through a holographic notch filter and collected via a CCD
us
camera.
an
2.4. DFT calculations
DFT calculations on Raman frequencies and assignments for the six Au atom cluster models were
M
performed with B3LYP/LanL2DZ functional/basis set using Gaussian 09 software [23]. The solvent effect caused by water molecules was considered by using the polarized continuum model (PCM)
d
implemented in Gaussian 09 software. Our PCM model recognized the aqueous environment, where all
te
the experiments were performed. Regarding the possibility of a parallel orientation, the geometry of 16
Ac ce p
Au atom cluster was optimized with B3LYP/LanL2DZ functional/basis set. The binding structures of GF to the gold cluster were obtained while the geometry of gold cluster was fixed.
2.5. Cell culture methods and cytotoxicity A549 (American type cell culture (ATCC), chronic lymphocytic chronic (CCL)-185), NCI-H460 (ATCC human tumor bank (HTB)-177, National Cancer Institute), and NCI-H1975 (ATCC thrombocytopenia (TCP)-1016, National Cancer Institute) were kindly provided from the Seoul National University. These are assumed to be GF resistant-cancer cell lines [12]. They were grown on Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% FBS and antibiotics at 37 °C in a 5% CO2 atmosphere incubator. RPMI and fetal bovine serum were obtained from WelGene 5
Page 5 of 23
(Daegu, Korea). The lung cancer cells were plated at a concentration of 1 × 106 cells per dish on a SPL 100 mm diameter cell culture dish (Seoul, Korea) containing growth medium. AuNPs were added and the cells were incubated at 37°C in 5 % CO2. After 48 h, the cells were harvested and washed with Dulbecco`s phosphate buffered saline solution. A cell viability test was performed using a Donjindo
ip t
CCK-8 kit. A Tecan Model F300 microplate reader was used to measure the absorbance change.
cr
3. Results and discussion
us
3.1. Molecular structure and DFT calculations
Fig. 1 illustrates the molecular structure of GF, which mainly consists of aromatic quinazoline and
an
phenyl rings. We expect that GF may adsorb on the AuNPs via the nitrogen atoms of the quinazoline ring [22]. Considering that most strong SERS bands can be ascribed to the quinazoline ring, we may
M
assume that GF may bind on Au via its nitrogen atoms. It is recognized that a cluster of just six atoms may be insufficient to explain the realistic adsorption behaviors of GF on AuNPs. Unlike erlotinib with
d
a strong anchoring unit of the acetylenic group [22], GF may weakly bind on AuNPs with its
te
quinazoline ring and lead to a rather flat bound geometry. Our DFT calculations of GF on 16 Au atom
Ac ce p
predicted that the binding energy would be 11.69 kcal/mol, which would be quite comparable to those of the standing geometries despite the difference of the Au substrate sizes. Although not shown here, the ν(CH) band at ~3050 cm-1, an indicator of the upright geometry, was barely identified, suggesting a rather flat geometry [24]. Considering that the aliphatic linker is distant from the quinazoline ring, the conformation variance would not affect the coordination at the surface. Based on DFT calculations of energetic stabilities using the PCM model, the N1 nitrogen atom position in the quinazoline ring of GF is predicted to be more stable in binding with the Au cluster atoms than those of the N3 position by 4.75 kcal/mol (summarized in Table 1). According to the Boltzmann distribution formula, the population of the adsorbed states with the energetic difference of 4.75 kcal/mol, may be calculated to be as low as ~0.03 %. The bond length of the N1 atom was calculated as 2.20 Å, which was shorter than 2.25 Å in the case of the N3 atom. 6
Page 6 of 23
3.2. UV-Vis absorption spectra of GF on AuNPs and loading efficiency The UV absorption band at 330 nm may be ascribed to the aromatic ring mode of GF. The loading efficiency of GF at the initial concentration of 10-5 M was estimated as 26-46% by subtracting the
ip t
unbound GF in the supernatant solution from the absorbance measurements. Depending on the initial concentrations of GF and solvents, the loading efficiencies appeared to be calculated slightly differently.
cr
The surface plasmon band could be ascribed to a collective motion of electrons on the surface of AuNPs
us
in aqueous colloidal solutions. The surface plasmon band redshifted after the adsorption of GF on AuNPs. After the assembly of antibodies, the UV-Vis spectra appeared to return almost to the original
an
positions of the pristine AuNPs owing to the extended interparticle separation via the PEG linker. Although not shown here, the infrared spectrum also indicated protein conjugation, showing the amide
M
vibrational bands at 1314 and 1658 cm-1. The TEM images in Fig. 2(b) suggest a slightly aggregated structure of AuNP-GF conjugates. The images on the right show an aggregated structure. In order to
Ac ce p
3.3. Raman spectra
te
d
better understand the interfacial phenomena of GF on AuNPs, we performed Raman spectroscopy.
Fig. 3(i) shows Raman spectra before and after the adsorption of GF on AuNPs. The spectral changes were not found to be dramatically different after adsorption of the solid GF on the solution phase AuNPs. Despite the drug adsorption on the AuNPs, the intrinsic SERS intensity of GF appeared to be rather weak in comparison to other simpler aromatic adsorbates such as thiophenol. The vibrational -1 bands between 1300 and 1425 cm can be ascribed to the quinazoline ring modes. These bands appeared
to change significantly upon adsorption on AuNPs, suggesting interaction with the surfaces. One of the strongest bands at 1337 cm-1 appeared to redshift to 1301 cm-1 upon adsorption on Au. AuNPs were covered and stabilized with the organic citrate anions. Tripeptides with aliphatic chains do not have any strong Raman bands even at their concentrations higher than the aromatic drug compound of GF. The comparative Raman spectra of 2 mM GSH on AuNPs and pristine AuNPs showing no strong SERS 7
Page 7 of 23
bands were also compared in Fig. 3(ii). Spectral data with the appropriate vibrational assignments for GF based on the DFT calculations are summarized in Table 2. Concentration-dependent spectra -5 indicated that the SERS intensities would show their maximum values at ~10 M of GF as shown in Fig.
3(ii) and (iii). This concentration of ~10-5 M can correspond to full-monolayer coverage of aromatic
ip t
adsorbates on AuNPs. Fig. 4(a) and (b) shows that GF may detach easily in 2 mM glutathione (GSH) solution but not in cell culture medium solution. The concentration of the cell culture media solution
cr
was adjusted by the previously reported method [25]. This result suggests that GF may be released
us
inside cells but not by the cell culture media solution after the endocytosis of the AuNP conjugates.
an
3.4. Cellular uptake
Fig. 5(a) shows TEM images of GF-assembled AuNPs in A549 cells after the uptake. The arrows
M
indicate the locations of internalized NPs. We found that GF-assembled AuNPs were internalized in either endosomal or lysosomal compartments indicated by the arrow. Fig. 5(b) of the DFM image also
d
exhibited the internalized AuNPs inside cells. Although not shown here, we could not observe any
te
discernible GF Raman peaks inside cells, presumably owing to the intracellular release of GF via
Ac ce p
glutathione as in the previous report [25]. Considering that GF would release in cytosols via intracellular GSH, the endosomal encapsulation may not affect its efficacies significantly. In order to check the potential application of AuNP-GF conjugates in drug delivery, we performed a cell viability test.
3.5. Cytotoxicity of GF-assembled AuNPs in A549, NCI-H460, and NCI-H1975 cells A549, NCI-H460, and NCI-H1975 cells are known to have resistance to GF owing to the mutation in EGFR [11,12]. Fig. 6 shows the viability of A549, NCI-H460, and NCI-H1975 cells in 5-100 nM GF. We have chosen a GF-resistant cell to investigate the efficacies of the AuNPs-GF-antibody. The used AuNPs did not to affect the cell viabilities significantly as in the previous report [26]. We have to mention that only GF-AuNP conjugates without any targeting moiety showed similar cell viability 8
Page 8 of 23
reduction to free GF. Introducing the antibody was thus beneficial to reduce cell viability. It was found that cell viability decreased by 30~80 % after assembling the GF-AuNPs-antibody than those that used only free drugs at the concentrations of 0.05 and 0.1 μM for the three lung cancer cell lines. We plan to utilize our method as a potentially useful drug delivery system to enhance the efficacy of treating lung
ip t
cancer cells.
cr
4. Conclusions
us
Interfacial structures of GF on AuNPs were examined mainly by Raman spectroscopy. DFT calculations predicted plausible adsorption structures on Au cluster atoms. A cytotoxicity test indicated
an
that a complex with the targeting antibody could damage lung cancer cell lines more effectively. Cell viability appeared to decrease by 30-80 % after treating antibody-conjugated AuNPs with GF
M
concentrations of 0.05 and 0.1 μM, if compared to using only free GF. Our results suggest that AuNPs
Ac ce p
Acknowledgements
te
d
can be successfully employed for conjugating the TKI inhibitor of GF.
We would like to thank Semi Kim for her help on TEM measurements. SWJ, Prof. So Yeong Lee, and KL thank Dr. Kyong-Ah Yoon for their helpful comments on the EGFR mutations. Prof. K. Cho ackno wledges the support by the Human Resources Development of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea government Ministry of Trade, Industry & Energy (No. 20124030200070). Prof. D. Kim acknowledges support from the Korea government (MEST) No. 2011-0031496. Prof. J. Choo acknowledges that the Nano Material Technology Development Program supported this work through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology (grant number 2012035286).
9
Page 9 of 23
References [1] J.L. Brash and P.W. Wojciechowski (Eds.), Interfacial Phenomena and Bioproducts, Marcel Dekker Inc., 1996, New York. [2] P. Joshi, S. Chakraborti, J.E. Ramirez-Vick, Z.A. Ansari, V. Shanker, P. Chakrabarti, S.P. Singh,
ip t
Colloids Surf. B 95 (2012) 195.
[3] H. Liu, M. Shen, J. Zhao, R. Guo, X. Cao, G. Zhang, X. Shi, Colloids Surf. B 94 (2012) 58.
cr
[4] A.F. de Faria, D.S.T. Martinez, S.M.M. Meira, A.C.M. de Moraes, A. Brandelli, A.G.S. Filho, O.L.
us
Alves, Colloids Surf. B 113 (2014) 115.
[5] R.K. Mohantya, S. Thennarasua, A.B. Mandal, Colloids Surf. B 114 (2014) 138.
an
[6] R.Y. Pelgrift, A.J. Friedman, Adv. Drug Deliv. Rev. 65 (2013) 1803.
[7] J.P. Almeida, E.R. Figueroa, R.A. Drezek, Nanomedicine 10 (2014) 503.
M
[8] S. Suarasan, M. Focsan, D. Maniu, S. Astilean, Colloids Surf. B 103 (2013) 475.
(2014) 276.
d
[9] A. Bhogale, N. Patel, J. Mariam, P.M. Dongre, A. Miotello, D.C. Kothari, Colloids Surf. B 113
te
[10] K. Tonigold, A. Groβ, J. Chem. Phys. 132 (2010) 224701.
Ac ce p
[11] S.V. Sharma, D.W. Bell, J. Settleman, D.A. Haber, Nat. Rev. Cancer. 7 (2007) 169. [12] M.D. To, C.E. Wong, A.N. Karnezis, R. Del Rosario, R. Di Lauro, A. Balmain, Nat. Genet. 40 (2008) 1240.
[13] H. Kimura, K. Kasahara, M. Kawaishi, H. Kunitoh, T. Tamura, B. Holloway, K. Nishio, Clin. Cancer Res. 12 (2006) 3915.
[14] T.J. Lynch, D.W. Bell, R. Sordella, S. Gurubhagavatula, R.A. Okimoto, B.W. Brannigan, P.L. Harris, S.M. Haserlat, J.G. Supko, F.G. Haluska, D.N. Louis, D.C. Christiani, J. Settleman, D.A. Haber, N. Eng. J. Med. 350 (2004) 2129. [15] W. Pao, M. Ladanyi. Clin. Cancer Res. 13 (2007) 4954.
10
Page 10 of 23
[16] K. Hoshi, H. Takakura, Y. Mitani, K. Tatsumi, N. Momiyama, Y. Ichikawa, S. Togo, T. Miyagi, Y. Kawai, Y. Kogo, T. Kikuchi, C. Kato, T. Arakawa, S. Uno, P.E. Cizdziel, A. Lezhava, N. Ogawa, Y. Hayashizaki, H. Shimada, Clin. Cancer Res. 13 (2007) 4974. [17] S. Kobayashi, T.J. Boggon, T. Dayaram, P.A. Jänne, O. Kocher, M. Meyerson, B.E. Johnson, M.J.
ip t
Eck, D.G. Tenen, B. Halmos, N. Engl. J. Med. 352 (2005) 786. [18] K.L. Mueller, Z-Q. Yang, R. Haddad, S.P. Ethier, J.L. Boerner, J. Mol. Signal. 5 (2010) 8.
cr
[19] J.G. Paez, P.A. Jänne, J.C. Lee, S. Tracy, H. Greulich, S. Gabriel, P. Herman, F.J. Kaye, N.
Meyerson, Science 304 (2004) 1497.
an
[20] P.C. Lee, D. Meisel, J. Phys. Chem. 86 (1982) 3391.
us
Lindeman, T.J. Boggon, K. Naoki, H. Sasaki, Y. Fujii, M.J. Eck, W.R. Sellers, B.E. Johnson, M.
[21] S. Lee, S. Kim, J. Choo, S.Y. Shin, Y.H. Lee, H.Y. Choi, S. Ha, K. Kang, C.H. Oh, Anal. Chem. 79
M
(2007) 916.
[22] A.T.N. Lam, J. Yoon, E-O. Ganbold, D.K. Singh, D. Kim, K-H. Cho, S.J. Son, J. Choo, S.Y. Lee, S.
d
Kim, S-W. Joo, J. Colloids Interface Sci. 425 (2014) 96.
te
[23] Gaussian 09, Revision D.01, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Ac ce p
Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, and D. J. Fox, Gaussian, Inc., Wallingford CT, 2009. [24] S-W. Joo, Y.S. Kim, Colloids Surf. A 234 (2004) 117. 11
Page 11 of 23
[25] K-S. Ock, E-O. Ganbold, J. Park, K. Cho, S-W. Joo, S.Y. Lee, Analyst 137 (2012) 2852. [26] S.Y. Choi, S. Jeong, S.H. Jang, J. Park, J.H. Park, K.-S. Ock, S.Y. Lee, S-W. Joo, Toxicol. In Vitro.
Ac ce p
te
d
M
an
us
cr
ip t
26 (2012) 229.
12
Page 12 of 23
Figure Captions
Fig. 1. (a) Molecular structure of GF. Optimized structures of GF with (b) N3 and (c) N1 binding onto the six Au atoms. (d) A parallel geometry of GF on 16 atom Au surfaces.
Fig. 2. (a) UV spectra of free and supernatant GF. UV-Vis spectra after conjugating GF with the
ip t
antibody and applying 2 mM GSH on AuNPs. (b) TEM images of GF-coated AuNPs. The
cr
scale bars in the left and right images are 200 nm and 5 nm, respectively.
Fig. 3. (i) (a) Ordinary Raman (OR) in the solid state and (b) SERS spectra after GF adsorption on
us
AuNPs. (ii) Concentration-dependent SERS spectra of GF on AuNPs and (iii) intensity plots of the bands at ~690 cm-1. SERS spectra of pristine AuNPs and 2 mM GSH are also listed for
an
comparison. The error bars show the standard deviations of the three independent measurements.
M
Fig. 4. (a) SERS spectra of GF on AuNPs in cell culture media of 0–100 % according to the procedures
te
d
described in reference [22]. (b) Time-dependent SERS spectra after treating 2 mM GSH.
Fig. 5. (a) TEM and (b) DFM images of GF-assembled AuNPs in A549 cells after the endocytosis. The
Ac ce p
scale bars are 2 μm and 15 μm, respectively. The red arrows indicate the locations of internalized AuNPs.
Fig. 6. Cell viability tests of free GF and AuNP-GF conjugates attached with the antibody for (a) A549, (b) NCI-H460, and (c) NCI-H1975 lung cancer cells after 48 h. The error bars show the standard deviations of three independent measurements.
13
Page 13 of 23
(c)
(d)
Ac ce p
te
d
M
an
us
(b)
cr
ip t
(a)
Fig. 1. (a) Molecular structure of GF. Optimized structures of GF with (b) N3 and (c) N1 binding onto the six Au atoms. (d) A parallel geometry of GF on 16 atom Au surfaces.
14
Page 14 of 23
(a) 0.4
Au N P s-G F An tibo d y
GF
cr
2 mM GSH
us
0.1 S u p ern atan t
0.0
400
500 600 700 800 W avelength (nm )
900
M
300
ip t
528
330
0.2
an
Absorbance
0.3
AuN P s-G F
Ac ce p
te
d
(b)
Fig. 2. (a) UV spectra of free and supernatant GF. UV-Vis spectra after conjugating GF with the antibody and applying 2 mM GSH on AuNPs. (b) TEM images of GF-coated AuNPs. The scale bars in the left and right images are 200 nm and 5 nm, respectively.
15
Page 15 of 23
1337
ip t cr
690 774
5000
(a)
1152 1204 1265 1301 1386 1401 1514 1576 1608
10000
1398 1425 1513 1578 1608
1217 1252
774
15000
257
(b)
0
500
us
Raman intensity (Arbitr. Unit)
(i)
20000
1000
1500
2000
an
Raman Shift (cm -1 )
(iii)
M
(ii)
2500
d
10-4 M
10000 8000 6000 4000 2000 0
500
1000 1500 2000 Raman Shift (cm-1)
3000
5x10-5 10-5 5x10-6 10-6 5x10-7 10-7 M AuNPs AuNPs -GSH
2500
Relative Intensity
te
12000
Ac ce p
Raman intensity (Arbitr. Unit)
14000
2000 1000 0 -7
10
-6
10 Concentration(M)
-5
10
Fig. 3. (i) (a) Ordinary Raman (OR) in the solid state and (b) SERS spectra after GF adsorption on AuNPs. (ii) Concentration-dependent SERS spectra of GF on AuNPs and (iii) intensity plots of the bands at ~690 cm-1. SERS spectra of pristine AuNPs and 2 mM GSH are also listed for comparison. The error bars show the standard deviations of the three independent measurements. 16
Page 16 of 23
6000 5000
ip t
4000
cr
3000 2000
us
1000
2500
d
M
an
1000 1500 2000 -1 Raman Shift (cm )
te
Raman Intensity (Arbitr. Unit)
500
Ac ce p
(b)
Raman Intensity (Arbitr. Unit)
(a)
0% Media 33% Media 66% Media 100 % media
7000
500
1000 1500 2000 Raman Shift (cm -1 )
2500
Fig. 4. (a) SERS spectra of GF on AuNPs in cell culture media of 0–100 % according to the procedures described in reference [22]. (b) Time-dependent SERS spectra after treating 2 mM GSH.
17
Page 17 of 23
(b)
Ac ce p
te
d
M
an
us
cr
ip t
(a)
Fig. 5. (a) TEM and (b) DFM images of GF-assembled AuNPs in A549 cells after the endocytosis. The scale bars are 2 μm and 15 μm, respectively. The red arrows indicate the locations of internalized AuNPs.
18
Page 18 of 23
us
cr
ip t
(a)
(c)
Ac ce p
te
d
M
an
(b)
Fig. 6. Cell viability tests of free GF and AuNP-GF conjugates attached with the antibody for (a) A549, (b) NCI-H460, and (c) NCI-H1975 lung cancer cells after 48 h. The error bars show the standard deviations of three independent measurements. 19
Page 19 of 23
Table 1. Binding energies of the six and sixteen gold atoms with GF and the corresponding bond distances and angles using the PCM model. ___________________________________________________________________________________ Configurations
B.E. (kcal/mol)
Distance (Å)
13.11
Au---N3
GF +Au6---N1
17.86
Au---N1
GF + Au16 parallel
11.69
2.25
us
cr
GF +Au6---N3
ip t
___________________________________________________________________________________
2.20
4.02
Au---N1
4.07
an
Au---N3
___________________________________________________________________________________
Ac ce p
te
d
M
Au6: optimized energy= -812.9800 au. GF optimized energy with PCM model= -1412.0308 au.
20
Page 20 of 23
Table 2. Spectral data and vibrational assignments for GF. Au SERS
774
1150
ν(Au-N)
690
ν(phenyl)
774
ν(phenyl)
1152
ν(quinazoline)
ν(phenyl)+β(C-H)
1217
1204
1240
1252
1265
1357
1337
1301
1402
1398
1386
1409
1425
1401
1522
1513
1514
1582
1578
1576
1609
1608
1608
β(N-H)+ ν(C-N)
ν(quinazoline) ν(quinazoline)+β(CH2)
ν(quinazoline)+β(C-H)+β(CH2) ν(phenyl)+ν(C-N) ν(quinazoline) ν(phenyl)+ν(C-N)+β(N-H)
d
M
1232
ip t
770
257
cr
693
Assignment
us
OR
an
Cal.
te
Unit in cm-1. Abbreviations: Cal.: calculated values; : stretching; β: in-plane bending; γ: out-of-plane
Ac ce p
bending. The assignment is based on the present DFT calculation of Raman frequencies for the GF N1coordinated six Au atom cluster models with B3LYP/LanL2DZ functional/basis set. No scale factor was applied in the calculated vibrational positions. OR positions were listed after the baseline correction.
21
Page 21 of 23
GRAPHICAL ABSTRACT
of Contents Use
Only
Ac ce p
te
For Table
d
M
an
us
cr
ip t
Colloidal gold nanoparticle conjugates of gefitinib
22
Page 22 of 23
Highlight
Ac ce p
te
d
M
an
us
cr
ip t
• Gefitinib-gold colloidal nanoparticle complex was fabricated by self-assembly. •Surface-enhanced Raman scattering was used to check the adsorption behaviors of gefitinib. • Cytotoxicity test indicated that the complex could damage lung cancer cells effectively.
23
Page 23 of 23
S0927-7765(14)00444-5 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.08.021 COLSUB 6586
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
5-1-2014 15-8-2014 17-8-2014
Please cite this article as: A.T.N. Lam, J. Yoon, E.-O. Ganbold, D.K. Singh, D. Kim, K.-H. Cho, S.Y. Lee, J. Choo, K. Lee, S.-W. Joo, Colloidal gold nanoparticle conjugates of gefitinib, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.08.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
an
us
cr
ip t
Colloidal gold nanoparticle conjugates of gefitinib
a a a b b Anh Thu Ngoc Lam , Jinha Yoon , Erdene-Ochir Ganbold , Dheeraj K. Singh , Doseok Kim , Kwang-
a
d
M
Hwi Choc, So Yeong Leed, Jaebum Chooe, Kangtaek Leef, Sang-Woo Joo a ,*
Department of Chemistry, Soongsil University, Seoul 156-743 Korea Department of Physics, Sogang University Seoul 121-742 Korea
c
School of Systems Biomedical Science, Soongsil University, Sangdo-dong, Dongjak-gu, Seoul, Korea
d
School of Veterinay Science, Seoul National University, Seoul 156-743, Korea
e
Department of Bionano Engineering, Hanyang University, Sa-1-dong 1271, Ansan 426-791, South Korea
f
Ac ce p
te
b
Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Republic of Korea
*
Corresponding author E-mail address: [email protected]
1
Page 1 of 23
Abstract
Gefitinib (GF) is a US Food and Drug Administration-approved epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor for treating the lung cancers. We fabricated colloidal gold nanoparticle
ip t
(AuNP) conjugates of the GF anticancer drug by self-assembly to test their potency against A549, NCIH460, and NCI-H1975 lung cancer cells. GF adsorption on AuNP surfaces was examined by UV-Vis
cr
absorption spectra and surface-enhanced Raman scattering. Density functional theory calculations were
us
performed to estimate the energetic stabilities of the drug-AuNP composites. The N1 nitrogen atom of the quinazoline ring of GF was calculated to be more stable than the N3 in binding Au cluster atoms.
an
The internalizations of GF-coated AuNPs were examined by transmission electron and dark-field microscopy. A cell viability test of AuNP-GF conjugates with the EGFR antibody exhibited much
M
higher reductions than free GF for A549, NCI-H460, and NCI-H1975 lung cancer cells after treatment
te
d
for 48 h.
Ac ce p
Keywords: Lung cancer cells; Gold nanoparticle conjugates; Gefitinib; Cell viability; Surface-enhanced Raman scattering
2
Page 2 of 23
1. Introduction Recently, understanding and controlling interfacial phenomena in biotechnology has become increasingly important in both fundamental research and industry [1]. Noble metal nanoparticles (NPs) have been introduced as novel drug delivery systems for anticancer therapy [2-5]. Gold nanoparticles
ip t
(AuNPs) after binding with the drugs may overcome antibiotic drug resistance [6]. AuNPs are known to be less toxic and can possibly be used in a hospital environment for immunotherapy [7]. Surface-
cr
enhanced Raman scattering (SERS) has been used to examine the adsorption structures in bioconjugate
us
chemistry [8,9]. Quantum mechanical calculations can be helpful to better understand the adsorption structures of self-assembled monolayers on metal cluster atoms [10].
an
Lung cancer is one of the leading causes of cancer-related deaths worldwide [11]. Expressed epidermal growth factor receptor (EGFR) can be utilized in lung cancer as a prime candidate for the development
M
of targeted therapeutics. Targeting EGFR is thus one of the most promising strategies for lung cancer therapy. In association with EGFR, tyrosine kinase inhibitor (TKI) has emerged as an effective cure for
d
some patients [12-16]. An EGFR-targeting small-molecule inhibitor, gefitinib (GF), received approval
te
from the US Food and Drug Administration as treatment for lung cancer patients, because it specifically
Ac ce p
targets the tyrosine kinase activity of EGFR, is one of the most popular and commercially available drugs to treat lung cancers [17-19].
Despite previous studies of nanoparticle conjugates with anticancer drugs, there have been no reports on the interfacial behavior of GF on AuNPs. GF may coordinate with AuNP surfaces via the quinazoline ring. In this work, we fabricated an AuNP conjugate of GF by self-assembly. Density functional theory (DFT) calculations were introduced to predict the plausible geometries of GF on an Au atom cluster. The adsorption behavior of GF on AuNP surfaces was studied mainly by means of SERS. A cell viability test was performed to examine their potency against A549, NCI-H460, and NCIH1975 lung cancer cells. Our work may be potentially useful in the application of AuNP conjugates in lung cancer therapy.
3
Page 3 of 23
2. Experimental section 2.1. Chemicals HAuCl4 and citrate were purchased from Sigma-Aldrich (St. Louis, USA). GF and mouse monoclonal anti-EGFR antibody (catalogue number: Ab62, concentration 1.2 mg/mL) were purchased from Selleck
ip t
Chemicals (Houston, USA) and Abcam (Cambridge, UK), respectively. Dimethyl sulfoxide (DMSO) was purchased from Daejung (Siheung, Korea). The (ortho-pyridyl) disulfide-poly(ethylene glycol)-N-
cr
hydroxysuccinimidyl ester (OPSS-PEG-NHS) linker was purchased from Creative PEGWorks (Winston
us
Salem, USA).
an
2.2. Preparation of nanoparticles
Citrate-modified AuNPs were prepared according to the previous procedure [20]. The AuNP-GF
M
sample was prepared by adding 10 μL of GF to 1 mL of AuNPs via a self-assembly process. The mixture was centrifuged at 10000 rpm for 5 min. To estimate the loading efficiency of GF onto AuNPs,
d
the UV band of 1 mL of supernatant solution was compared with that of 1 mL of the precipitated
te
solution redispersed in DMSO. The loading efficiency of AuNPs-GF (10-5 M) in DMSO was estimated
Ac ce p
to be 26-46 %, depending on the solvents and concentrations. To attach the EGFR targeting moiety to AuNPs, we suspended 10 µL of antibody (1mg/mL) in 90 µL of NaHCO3 (pH 8.5) according to the previous report [21]. The OPSS-PEG-NHS linker was prepared with a concentration of 1mg/mL (1 mg of OPSS in 1 mL of NaHCO3 of pH 8.5). Then, 100 µL of antibody and 100 µL of OPSS-PEG-NHS in a ratio of 1:1 was mixed and maintained at 4 °C for approximately 3 h. Subsequently, 1mL of AuNP-GF was added to 200 µL of antibody-OPSS-PEG-NHS and maintained at 4 °C overnight. The mixture solution was then centrifuged at 10,000 rpm for 5 min. After collecting the precipitate, it was dissolved again in 1 mL of DMSO solution. AuNPs-GF-antibody was prepared with a GF concentration of 10-4 M.
2.3. Equipment and characterization methods
4
Page 4 of 23
This amount of AuNP conjugates was diluted to 10-5 M by DMSO solvent and used for the CCK experiment. The UV-Vis absorbance spectrum of the surface plasmon band from the AuNP solution was obtained using a Scinco Mecasys 3220 spectrophotometer. The size distributions of AuNPs were checked using either a JEOL JEM-3010 or JEM-1010 transmission electron microscope (TEM). Raman
ip t
spectra equipped with dark-field microscopy (DFM) were obtained using a Renishaw 1000 microRaman spectrometer as in the previous report [22]. The excitation wavelength was 632.8 nm. The spontaneous
cr
scattering from the sample was dispersed through a holographic notch filter and collected via a CCD
us
camera.
an
2.4. DFT calculations
DFT calculations on Raman frequencies and assignments for the six Au atom cluster models were
M
performed with B3LYP/LanL2DZ functional/basis set using Gaussian 09 software [23]. The solvent effect caused by water molecules was considered by using the polarized continuum model (PCM)
d
implemented in Gaussian 09 software. Our PCM model recognized the aqueous environment, where all
te
the experiments were performed. Regarding the possibility of a parallel orientation, the geometry of 16
Ac ce p
Au atom cluster was optimized with B3LYP/LanL2DZ functional/basis set. The binding structures of GF to the gold cluster were obtained while the geometry of gold cluster was fixed.
2.5. Cell culture methods and cytotoxicity A549 (American type cell culture (ATCC), chronic lymphocytic chronic (CCL)-185), NCI-H460 (ATCC human tumor bank (HTB)-177, National Cancer Institute), and NCI-H1975 (ATCC thrombocytopenia (TCP)-1016, National Cancer Institute) were kindly provided from the Seoul National University. These are assumed to be GF resistant-cancer cell lines [12]. They were grown on Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% FBS and antibiotics at 37 °C in a 5% CO2 atmosphere incubator. RPMI and fetal bovine serum were obtained from WelGene 5
Page 5 of 23
(Daegu, Korea). The lung cancer cells were plated at a concentration of 1 × 106 cells per dish on a SPL 100 mm diameter cell culture dish (Seoul, Korea) containing growth medium. AuNPs were added and the cells were incubated at 37°C in 5 % CO2. After 48 h, the cells were harvested and washed with Dulbecco`s phosphate buffered saline solution. A cell viability test was performed using a Donjindo
ip t
CCK-8 kit. A Tecan Model F300 microplate reader was used to measure the absorbance change.
cr
3. Results and discussion
us
3.1. Molecular structure and DFT calculations
Fig. 1 illustrates the molecular structure of GF, which mainly consists of aromatic quinazoline and
an
phenyl rings. We expect that GF may adsorb on the AuNPs via the nitrogen atoms of the quinazoline ring [22]. Considering that most strong SERS bands can be ascribed to the quinazoline ring, we may
M
assume that GF may bind on Au via its nitrogen atoms. It is recognized that a cluster of just six atoms may be insufficient to explain the realistic adsorption behaviors of GF on AuNPs. Unlike erlotinib with
d
a strong anchoring unit of the acetylenic group [22], GF may weakly bind on AuNPs with its
te
quinazoline ring and lead to a rather flat bound geometry. Our DFT calculations of GF on 16 Au atom
Ac ce p
predicted that the binding energy would be 11.69 kcal/mol, which would be quite comparable to those of the standing geometries despite the difference of the Au substrate sizes. Although not shown here, the ν(CH) band at ~3050 cm-1, an indicator of the upright geometry, was barely identified, suggesting a rather flat geometry [24]. Considering that the aliphatic linker is distant from the quinazoline ring, the conformation variance would not affect the coordination at the surface. Based on DFT calculations of energetic stabilities using the PCM model, the N1 nitrogen atom position in the quinazoline ring of GF is predicted to be more stable in binding with the Au cluster atoms than those of the N3 position by 4.75 kcal/mol (summarized in Table 1). According to the Boltzmann distribution formula, the population of the adsorbed states with the energetic difference of 4.75 kcal/mol, may be calculated to be as low as ~0.03 %. The bond length of the N1 atom was calculated as 2.20 Å, which was shorter than 2.25 Å in the case of the N3 atom. 6
Page 6 of 23
3.2. UV-Vis absorption spectra of GF on AuNPs and loading efficiency The UV absorption band at 330 nm may be ascribed to the aromatic ring mode of GF. The loading efficiency of GF at the initial concentration of 10-5 M was estimated as 26-46% by subtracting the
ip t
unbound GF in the supernatant solution from the absorbance measurements. Depending on the initial concentrations of GF and solvents, the loading efficiencies appeared to be calculated slightly differently.
cr
The surface plasmon band could be ascribed to a collective motion of electrons on the surface of AuNPs
us
in aqueous colloidal solutions. The surface plasmon band redshifted after the adsorption of GF on AuNPs. After the assembly of antibodies, the UV-Vis spectra appeared to return almost to the original
an
positions of the pristine AuNPs owing to the extended interparticle separation via the PEG linker. Although not shown here, the infrared spectrum also indicated protein conjugation, showing the amide
M
vibrational bands at 1314 and 1658 cm-1. The TEM images in Fig. 2(b) suggest a slightly aggregated structure of AuNP-GF conjugates. The images on the right show an aggregated structure. In order to
Ac ce p
3.3. Raman spectra
te
d
better understand the interfacial phenomena of GF on AuNPs, we performed Raman spectroscopy.
Fig. 3(i) shows Raman spectra before and after the adsorption of GF on AuNPs. The spectral changes were not found to be dramatically different after adsorption of the solid GF on the solution phase AuNPs. Despite the drug adsorption on the AuNPs, the intrinsic SERS intensity of GF appeared to be rather weak in comparison to other simpler aromatic adsorbates such as thiophenol. The vibrational -1 bands between 1300 and 1425 cm can be ascribed to the quinazoline ring modes. These bands appeared
to change significantly upon adsorption on AuNPs, suggesting interaction with the surfaces. One of the strongest bands at 1337 cm-1 appeared to redshift to 1301 cm-1 upon adsorption on Au. AuNPs were covered and stabilized with the organic citrate anions. Tripeptides with aliphatic chains do not have any strong Raman bands even at their concentrations higher than the aromatic drug compound of GF. The comparative Raman spectra of 2 mM GSH on AuNPs and pristine AuNPs showing no strong SERS 7
Page 7 of 23
bands were also compared in Fig. 3(ii). Spectral data with the appropriate vibrational assignments for GF based on the DFT calculations are summarized in Table 2. Concentration-dependent spectra -5 indicated that the SERS intensities would show their maximum values at ~10 M of GF as shown in Fig.
3(ii) and (iii). This concentration of ~10-5 M can correspond to full-monolayer coverage of aromatic
ip t
adsorbates on AuNPs. Fig. 4(a) and (b) shows that GF may detach easily in 2 mM glutathione (GSH) solution but not in cell culture medium solution. The concentration of the cell culture media solution
cr
was adjusted by the previously reported method [25]. This result suggests that GF may be released
us
inside cells but not by the cell culture media solution after the endocytosis of the AuNP conjugates.
an
3.4. Cellular uptake
Fig. 5(a) shows TEM images of GF-assembled AuNPs in A549 cells after the uptake. The arrows
M
indicate the locations of internalized NPs. We found that GF-assembled AuNPs were internalized in either endosomal or lysosomal compartments indicated by the arrow. Fig. 5(b) of the DFM image also
d
exhibited the internalized AuNPs inside cells. Although not shown here, we could not observe any
te
discernible GF Raman peaks inside cells, presumably owing to the intracellular release of GF via
Ac ce p
glutathione as in the previous report [25]. Considering that GF would release in cytosols via intracellular GSH, the endosomal encapsulation may not affect its efficacies significantly. In order to check the potential application of AuNP-GF conjugates in drug delivery, we performed a cell viability test.
3.5. Cytotoxicity of GF-assembled AuNPs in A549, NCI-H460, and NCI-H1975 cells A549, NCI-H460, and NCI-H1975 cells are known to have resistance to GF owing to the mutation in EGFR [11,12]. Fig. 6 shows the viability of A549, NCI-H460, and NCI-H1975 cells in 5-100 nM GF. We have chosen a GF-resistant cell to investigate the efficacies of the AuNPs-GF-antibody. The used AuNPs did not to affect the cell viabilities significantly as in the previous report [26]. We have to mention that only GF-AuNP conjugates without any targeting moiety showed similar cell viability 8
Page 8 of 23
reduction to free GF. Introducing the antibody was thus beneficial to reduce cell viability. It was found that cell viability decreased by 30~80 % after assembling the GF-AuNPs-antibody than those that used only free drugs at the concentrations of 0.05 and 0.1 μM for the three lung cancer cell lines. We plan to utilize our method as a potentially useful drug delivery system to enhance the efficacy of treating lung
ip t
cancer cells.
cr
4. Conclusions
us
Interfacial structures of GF on AuNPs were examined mainly by Raman spectroscopy. DFT calculations predicted plausible adsorption structures on Au cluster atoms. A cytotoxicity test indicated
an
that a complex with the targeting antibody could damage lung cancer cell lines more effectively. Cell viability appeared to decrease by 30-80 % after treating antibody-conjugated AuNPs with GF
M
concentrations of 0.05 and 0.1 μM, if compared to using only free GF. Our results suggest that AuNPs
Ac ce p
Acknowledgements
te
d
can be successfully employed for conjugating the TKI inhibitor of GF.
We would like to thank Semi Kim for her help on TEM measurements. SWJ, Prof. So Yeong Lee, and KL thank Dr. Kyong-Ah Yoon for their helpful comments on the EGFR mutations. Prof. K. Cho ackno wledges the support by the Human Resources Development of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea government Ministry of Trade, Industry & Energy (No. 20124030200070). Prof. D. Kim acknowledges support from the Korea government (MEST) No. 2011-0031496. Prof. J. Choo acknowledges that the Nano Material Technology Development Program supported this work through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology (grant number 2012035286).
9
Page 9 of 23
References [1] J.L. Brash and P.W. Wojciechowski (Eds.), Interfacial Phenomena and Bioproducts, Marcel Dekker Inc., 1996, New York. [2] P. Joshi, S. Chakraborti, J.E. Ramirez-Vick, Z.A. Ansari, V. Shanker, P. Chakrabarti, S.P. Singh,
ip t
Colloids Surf. B 95 (2012) 195.
[3] H. Liu, M. Shen, J. Zhao, R. Guo, X. Cao, G. Zhang, X. Shi, Colloids Surf. B 94 (2012) 58.
cr
[4] A.F. de Faria, D.S.T. Martinez, S.M.M. Meira, A.C.M. de Moraes, A. Brandelli, A.G.S. Filho, O.L.
us
Alves, Colloids Surf. B 113 (2014) 115.
[5] R.K. Mohantya, S. Thennarasua, A.B. Mandal, Colloids Surf. B 114 (2014) 138.
an
[6] R.Y. Pelgrift, A.J. Friedman, Adv. Drug Deliv. Rev. 65 (2013) 1803.
[7] J.P. Almeida, E.R. Figueroa, R.A. Drezek, Nanomedicine 10 (2014) 503.
M
[8] S. Suarasan, M. Focsan, D. Maniu, S. Astilean, Colloids Surf. B 103 (2013) 475.
(2014) 276.
d
[9] A. Bhogale, N. Patel, J. Mariam, P.M. Dongre, A. Miotello, D.C. Kothari, Colloids Surf. B 113
te
[10] K. Tonigold, A. Groβ, J. Chem. Phys. 132 (2010) 224701.
Ac ce p
[11] S.V. Sharma, D.W. Bell, J. Settleman, D.A. Haber, Nat. Rev. Cancer. 7 (2007) 169. [12] M.D. To, C.E. Wong, A.N. Karnezis, R. Del Rosario, R. Di Lauro, A. Balmain, Nat. Genet. 40 (2008) 1240.
[13] H. Kimura, K. Kasahara, M. Kawaishi, H. Kunitoh, T. Tamura, B. Holloway, K. Nishio, Clin. Cancer Res. 12 (2006) 3915.
[14] T.J. Lynch, D.W. Bell, R. Sordella, S. Gurubhagavatula, R.A. Okimoto, B.W. Brannigan, P.L. Harris, S.M. Haserlat, J.G. Supko, F.G. Haluska, D.N. Louis, D.C. Christiani, J. Settleman, D.A. Haber, N. Eng. J. Med. 350 (2004) 2129. [15] W. Pao, M. Ladanyi. Clin. Cancer Res. 13 (2007) 4954.
10
Page 10 of 23
[16] K. Hoshi, H. Takakura, Y. Mitani, K. Tatsumi, N. Momiyama, Y. Ichikawa, S. Togo, T. Miyagi, Y. Kawai, Y. Kogo, T. Kikuchi, C. Kato, T. Arakawa, S. Uno, P.E. Cizdziel, A. Lezhava, N. Ogawa, Y. Hayashizaki, H. Shimada, Clin. Cancer Res. 13 (2007) 4974. [17] S. Kobayashi, T.J. Boggon, T. Dayaram, P.A. Jänne, O. Kocher, M. Meyerson, B.E. Johnson, M.J.
ip t
Eck, D.G. Tenen, B. Halmos, N. Engl. J. Med. 352 (2005) 786. [18] K.L. Mueller, Z-Q. Yang, R. Haddad, S.P. Ethier, J.L. Boerner, J. Mol. Signal. 5 (2010) 8.
cr
[19] J.G. Paez, P.A. Jänne, J.C. Lee, S. Tracy, H. Greulich, S. Gabriel, P. Herman, F.J. Kaye, N.
Meyerson, Science 304 (2004) 1497.
an
[20] P.C. Lee, D. Meisel, J. Phys. Chem. 86 (1982) 3391.
us
Lindeman, T.J. Boggon, K. Naoki, H. Sasaki, Y. Fujii, M.J. Eck, W.R. Sellers, B.E. Johnson, M.
[21] S. Lee, S. Kim, J. Choo, S.Y. Shin, Y.H. Lee, H.Y. Choi, S. Ha, K. Kang, C.H. Oh, Anal. Chem. 79
M
(2007) 916.
[22] A.T.N. Lam, J. Yoon, E-O. Ganbold, D.K. Singh, D. Kim, K-H. Cho, S.J. Son, J. Choo, S.Y. Lee, S.
d
Kim, S-W. Joo, J. Colloids Interface Sci. 425 (2014) 96.
te
[23] Gaussian 09, Revision D.01, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Ac ce p
Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, and D. J. Fox, Gaussian, Inc., Wallingford CT, 2009. [24] S-W. Joo, Y.S. Kim, Colloids Surf. A 234 (2004) 117. 11
Page 11 of 23
[25] K-S. Ock, E-O. Ganbold, J. Park, K. Cho, S-W. Joo, S.Y. Lee, Analyst 137 (2012) 2852. [26] S.Y. Choi, S. Jeong, S.H. Jang, J. Park, J.H. Park, K.-S. Ock, S.Y. Lee, S-W. Joo, Toxicol. In Vitro.
Ac ce p
te
d
M
an
us
cr
ip t
26 (2012) 229.
12
Page 12 of 23
Figure Captions
Fig. 1. (a) Molecular structure of GF. Optimized structures of GF with (b) N3 and (c) N1 binding onto the six Au atoms. (d) A parallel geometry of GF on 16 atom Au surfaces.
Fig. 2. (a) UV spectra of free and supernatant GF. UV-Vis spectra after conjugating GF with the
ip t
antibody and applying 2 mM GSH on AuNPs. (b) TEM images of GF-coated AuNPs. The
cr
scale bars in the left and right images are 200 nm and 5 nm, respectively.
Fig. 3. (i) (a) Ordinary Raman (OR) in the solid state and (b) SERS spectra after GF adsorption on
us
AuNPs. (ii) Concentration-dependent SERS spectra of GF on AuNPs and (iii) intensity plots of the bands at ~690 cm-1. SERS spectra of pristine AuNPs and 2 mM GSH are also listed for
an
comparison. The error bars show the standard deviations of the three independent measurements.
M
Fig. 4. (a) SERS spectra of GF on AuNPs in cell culture media of 0–100 % according to the procedures
te
d
described in reference [22]. (b) Time-dependent SERS spectra after treating 2 mM GSH.
Fig. 5. (a) TEM and (b) DFM images of GF-assembled AuNPs in A549 cells after the endocytosis. The
Ac ce p
scale bars are 2 μm and 15 μm, respectively. The red arrows indicate the locations of internalized AuNPs.
Fig. 6. Cell viability tests of free GF and AuNP-GF conjugates attached with the antibody for (a) A549, (b) NCI-H460, and (c) NCI-H1975 lung cancer cells after 48 h. The error bars show the standard deviations of three independent measurements.
13
Page 13 of 23
(c)
(d)
Ac ce p
te
d
M
an
us
(b)
cr
ip t
(a)
Fig. 1. (a) Molecular structure of GF. Optimized structures of GF with (b) N3 and (c) N1 binding onto the six Au atoms. (d) A parallel geometry of GF on 16 atom Au surfaces.
14
Page 14 of 23
(a) 0.4
Au N P s-G F An tibo d y
GF
cr
2 mM GSH
us
0.1 S u p ern atan t
0.0
400
500 600 700 800 W avelength (nm )
900
M
300
ip t
528
330
0.2
an
Absorbance
0.3
AuN P s-G F
Ac ce p
te
d
(b)
Fig. 2. (a) UV spectra of free and supernatant GF. UV-Vis spectra after conjugating GF with the antibody and applying 2 mM GSH on AuNPs. (b) TEM images of GF-coated AuNPs. The scale bars in the left and right images are 200 nm and 5 nm, respectively.
15
Page 15 of 23
1337
ip t cr
690 774
5000
(a)
1152 1204 1265 1301 1386 1401 1514 1576 1608
10000
1398 1425 1513 1578 1608
1217 1252
774
15000
257
(b)
0
500
us
Raman intensity (Arbitr. Unit)
(i)
20000
1000
1500
2000
an
Raman Shift (cm -1 )
(iii)
M
(ii)
2500
d
10-4 M
10000 8000 6000 4000 2000 0
500
1000 1500 2000 Raman Shift (cm-1)
3000
5x10-5 10-5 5x10-6 10-6 5x10-7 10-7 M AuNPs AuNPs -GSH
2500
Relative Intensity
te
12000
Ac ce p
Raman intensity (Arbitr. Unit)
14000
2000 1000 0 -7
10
-6
10 Concentration(M)
-5
10
Fig. 3. (i) (a) Ordinary Raman (OR) in the solid state and (b) SERS spectra after GF adsorption on AuNPs. (ii) Concentration-dependent SERS spectra of GF on AuNPs and (iii) intensity plots of the bands at ~690 cm-1. SERS spectra of pristine AuNPs and 2 mM GSH are also listed for comparison. The error bars show the standard deviations of the three independent measurements. 16
Page 16 of 23
6000 5000
ip t
4000
cr
3000 2000
us
1000
2500
d
M
an
1000 1500 2000 -1 Raman Shift (cm )
te
Raman Intensity (Arbitr. Unit)
500
Ac ce p
(b)
Raman Intensity (Arbitr. Unit)
(a)
0% Media 33% Media 66% Media 100 % media
7000
500
1000 1500 2000 Raman Shift (cm -1 )
2500
Fig. 4. (a) SERS spectra of GF on AuNPs in cell culture media of 0–100 % according to the procedures described in reference [22]. (b) Time-dependent SERS spectra after treating 2 mM GSH.
17
Page 17 of 23
(b)
Ac ce p
te
d
M
an
us
cr
ip t
(a)
Fig. 5. (a) TEM and (b) DFM images of GF-assembled AuNPs in A549 cells after the endocytosis. The scale bars are 2 μm and 15 μm, respectively. The red arrows indicate the locations of internalized AuNPs.
18
Page 18 of 23
us
cr
ip t
(a)
(c)
Ac ce p
te
d
M
an
(b)
Fig. 6. Cell viability tests of free GF and AuNP-GF conjugates attached with the antibody for (a) A549, (b) NCI-H460, and (c) NCI-H1975 lung cancer cells after 48 h. The error bars show the standard deviations of three independent measurements. 19
Page 19 of 23
Table 1. Binding energies of the six and sixteen gold atoms with GF and the corresponding bond distances and angles using the PCM model. ___________________________________________________________________________________ Configurations
B.E. (kcal/mol)
Distance (Å)
13.11
Au---N3
GF +Au6---N1
17.86
Au---N1
GF + Au16 parallel
11.69
2.25
us
cr
GF +Au6---N3
ip t
___________________________________________________________________________________
2.20
4.02
Au---N1
4.07
an
Au---N3
___________________________________________________________________________________
Ac ce p
te
d
M
Au6: optimized energy= -812.9800 au. GF optimized energy with PCM model= -1412.0308 au.
20
Page 20 of 23
Table 2. Spectral data and vibrational assignments for GF. Au SERS
774
1150
ν(Au-N)
690
ν(phenyl)
774
ν(phenyl)
1152
ν(quinazoline)
ν(phenyl)+β(C-H)
1217
1204
1240
1252
1265
1357
1337
1301
1402
1398
1386
1409
1425
1401
1522
1513
1514
1582
1578
1576
1609
1608
1608
β(N-H)+ ν(C-N)
ν(quinazoline) ν(quinazoline)+β(CH2)
ν(quinazoline)+β(C-H)+β(CH2) ν(phenyl)+ν(C-N) ν(quinazoline) ν(phenyl)+ν(C-N)+β(N-H)
d
M
1232
ip t
770
257
cr
693
Assignment
us
OR
an
Cal.
te
Unit in cm-1. Abbreviations: Cal.: calculated values; : stretching; β: in-plane bending; γ: out-of-plane
Ac ce p
bending. The assignment is based on the present DFT calculation of Raman frequencies for the GF N1coordinated six Au atom cluster models with B3LYP/LanL2DZ functional/basis set. No scale factor was applied in the calculated vibrational positions. OR positions were listed after the baseline correction.
21
Page 21 of 23
GRAPHICAL ABSTRACT
of Contents Use
Only
Ac ce p
te
For Table
d
M
an
us
cr
ip t
Colloidal gold nanoparticle conjugates of gefitinib
22
Page 22 of 23
Highlight
Ac ce p
te
d
M
an
us
cr
ip t
• Gefitinib-gold colloidal nanoparticle complex was fabricated by self-assembly. •Surface-enhanced Raman scattering was used to check the adsorption behaviors of gefitinib. • Cytotoxicity test indicated that the complex could damage lung cancer cells effectively.
23
Page 23 of 23
